Previous workers have utilized PEG or methoxy-PEG of low molecular weight (350 to 20,000, and typically about 5,000) attached to superoxide dismutase (SOD) and other proteins to obtain adducts demonstrating varying degrees of (a) increased serum persistence and (b) reduced immunogenicity. However, the extent of modification of protein groups with low molecular weight PEG or methoxy-PEG required to adequately attain both objectives (a) as well as (b), often leads to substantial losses in enzyme activity or biological activity. For example, Pyatak et al. (Res. Commun. Chem. Pathol. Pharmacol. 29, 113-127, 1980) showed that attaching 7 to 18 methoxy-PEG strands (MW 5000, each strand) to SOD produced adducts demonstrating a serum half life of about 25 hours in mice but which retained only 50%-60% of the native enzyme activity. Attaching fewer PEG strands of MW 5000 gave adducts of higher enzyme activity but reduced the serum persistence (to about 10 hours.)
Yoshimoto et al. (Jpn. J. Cancer Res. 77, 1264-1270, 1986) have attempted to solve the reduced activity problem observed with L-asparaginase, by modifying fewer protein groups using a chloro-triazine reagent substituted with two strands of methoxy-PEG, each strand having a molecular weight in the same range (5000 or less) employed by others. However, cyanuric chloride is toxic to animals and many workers have cautioned against introducing a triazine ring into an adduct used for pharmaceutical purposes, since this chemical moiety would be anticipated to be immunogenic. Yabuki and Iwashita (European Patent Application #86303058.1, filed 4/23/86) substituted PEG for methoxy-PEG, but in the same MW range employed by other workers, to prepare adducts which claim to be composed of SOD copolymers in which both ends of the PEG are attached to different SOD molecules. They report serum half-lives for these adducts of 4 to 8 hours. Other workers, including Lee and Sehon (Int. Archs. Allergy Appl. Immun., 56, 159-170, 1978), Lee and Sehon (ibid., 56, 193-206, 1978) and Mueller et al., (ibid., 68, 312-319, 1982 and ibid., 68, 320-325, 1982) had previously used PEG in the same MW range (6,000-20,000) as Yabuki and Iwashita to produce adducts which probably contained similar copolymer structures. Wie et al. (Int. Archs. Allergy Appl. Immun., 64, 84-99, 1981) and Lee et al. (ibid., 64, 100-114, 1981) specifically replaced PEG with methoxy-PEG in the 2000-20,000 MW range to avoid possible inter- and intramolecular linkages.